In healthy individuals, after eating, insulin secretion from beta cells takes place in two phases: a rapid but small first phase, and a delayed but sustained second phase. During this second phase, insulin is mobilized from the interior of the cell for secretion into the blood1. Microtubules, highly dynamic structural cellular elements, are necessary for this mobilization, but we do not fully understand how they coordinate this process. Specifically, we are investigating the role of a family of proteins which bind at the end of the microtubule. We believe that these microtubule end-binding proteins help coordinate insulin secretion by binding to the surface of the insulin granule, and in effect, tethering it to the microtubule. In this way, the insulin granule would preferentially associate near the edge of the cell, where it would be poised for secretion. Alternatively, the microtubule end-binding proteins might instead be acting to stabilize the microtubule, which frequently spontaneously disassembles. In this way, the insulin granule would have a steadier path from the interior of the cell. To investigate these hypotheses, we will perform a number of studies. First, because the microtubule end- binding proteins in beta cells are completely uncharacterized, we will first determine which proteins are expressed, both at the level of mRNA and protein. We will then determine how the end-binding proteins become associated with the insulin granule by isolating and identifying interacting proteins through mass spectrometry. Next, using isolated beta cells, we will ask what effect targeted depletion of the end-binding proteins and their interactors has on insulin secretion, both by: measuring the amount of insulin that is secreted;and by microscopically tracking the insulin granules as they are recruited from the interior of the beta cell to the surface. Finally, to enable formal dissection of this process, we will reconstitute insulin secretion using purified proteins, microtubules, and isolated insulin granules. These studies have the potential to help us understand how insulin secretion functions in normal individuals, and how it goes wrong in individuals with diabetes. Diabetes is fundamentally a disease of insulin secretion. Over time, the body becomes less sensitive to insulin, but symptoms of the disease only manifest when secretion from beta cells can no longer compensate. This is a serious problem;a recent study estimated that more than 44 million Americans will have diabetes within 25 years and that costs will triple to more than $300 billion. By studying these fundamental cellular processes, we seek to alleviate this looming burden. Reference List 1. Hou, J. C., Min, L. &Pessin, J. E. Insulin and IGFs (ed.), pp. 473-506 (Academic Press,2009). 2. Wang, Z. &Thurmond, D. C. Mechanisms of biphasic insulin-granule exocytosis - roles of the cytoskeleton, small GTPases and SNARE proteins. J Cell Sci 122, 893-903 (2009). 3. Huang, E. S., Basu, A., O'Grady, M. &Capretta, J. C. Projecting the future diabetes population size and related costs for the U.S. Diabetes Care 32, 2225-2229 (2009). PUBLIC HEALTH RELEVANCE: Insulin is normally efficiently transported from within specialized cells in the pancreas into the blood. When this process fails, diabetes occurs. We are studying the specifics of this transport process to better understand how this functions in normal individuals, which may reveal what fails in the setting of disease.